CN105524996A - Method for rapid detection of ichthyophthirius multifiliis in water body - Google Patents
Method for rapid detection of ichthyophthirius multifiliis in water body Download PDFInfo
- Publication number
- CN105524996A CN105524996A CN201610024984.6A CN201610024984A CN105524996A CN 105524996 A CN105524996 A CN 105524996A CN 201610024984 A CN201610024984 A CN 201610024984A CN 105524996 A CN105524996 A CN 105524996A
- Authority
- CN
- China
- Prior art keywords
- water body
- melonworm
- primer
- water
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for rapid detection of ichthyophthirius multifiliis in a water body. The method comprises the following steps: according to the size of an aquaculture water area, collecting a water sample; processing the water sample and extracting total DNA in the water body; conducting PCR amplification by taking known nucleotide sequences IC-ITS-F: 5'-GTTCCCCTTGAACGAGGAATTC-3' and IC-ITS-R: 5'-TTAGTTTCTTTTCCTCCGC-3' as primers, so that a PCR product is obtained; linking the PCR product to a vector and transferring to escherichia coli DH5 alpha for cloning; comparing sequencing results by virtue of DNAMAN6.0 software, and searching difference sites of the ichthyophthirius multifiliis with other similar species which can be detected and designing a nested PCR primer for the ichthyophthirius multifiliis; and conducting nested PCR on a primary PCR product by virtue of the primer; and completing detection if an electrophoresis detection result shows that strips can be amplified by the ichthyophthirius multifiliis. Through the application of the method, the existence of the ichthyophthirius multifiliis in the water body can be detected quite conveniently and effectively, so that an effective way is provided for timely water body treatment and for preventing the outbreak of the ichthyophthirius multifiliis; therefore, the method is high in practical value and is worthy of popularizing.
Description
Technical field
The present invention relates to biology field, the particularly collection of the medium and small melonworm DNA sample of water body, extraction, amplification and qualification aspect, specifically refer to the little melonworm method for quick of a kind of water body.
Background technology
Ichthyophthirius is in Protozoa, Ciliata, Hymenostomatida, recess section, Ichthyophthirius, it is a kind of universal parasite of freshwater fishes, in high-density aquiculture, cause fish overwhelming infection, cause large quantities of fish dead, cause tremendous economic to lose.
In order to effectively control the generation of this disease, domestic and international researcher has inquired into the manufacture to the killing effect of in vitro little melonworm, the different immune vaccine of little melonworm of the some biological characteristics of this disease pathogen, pathological characteristic, physical chemistry and herbal medicine.Except fundamental research the result of curative test be not effect undesirable be exactly that applicability is poor.Integrated culture experience and experimental result, the control strategy at present for ichthyophthiriasis should be relied mainly on prevention, and it is auxiliary for controlling.So understand the existence of the medium and small melonworm of water body in time, carrying out water body process in advance or transfer cultivation fish body in time, is the optimal path preventing ichthyophthiriasis from breaking out.
Summary of the invention
Content of the present invention there is situation in order to the medium and small melonworm of Timeliness coverage water body, there is provided one efficiently, fast, the little melonworm detection method of water body accurately, for judging that eventually through little melonworm DNA concentration the possibility breaking out ichthyophthiriasis lays the foundation, in order to instruct actual cultivation operation.
For achieving the above object, the invention provides a kind of medium and small melonworm method of rapid detection water body, comprise the following steps:
Step 1: according to Cultivated water size, gathers water sample;
Step 2: after being processed by water sample, extracts the STb gene in water body;
Step 3: utilize nucleotide sequence IC-ITS-F and IC-ITS-R to carry out pcr amplification as primer, obtain PCR primer 750bp, electrophoresis detection;
Step 4: above-mentioned PCR primer is cut glue and reclaim, proceed in bacillus coli DH 5 alpha after connection carrier and clone, and picking mono-clonal individuality as much as possible checks order;
Step 5: utilize DNAMAN6.0 software to compare to sequencing result, finds out the difference site between little melonworm and the sibling species recorded and designs at least 2 to the Chao Shi PCR primer for little melonworm;
Step 6: the PCR primer utilizing above-mentioned primer pair step 3 to obtain carries out Chao Shi PCR, knows that electrophoresis detection result shows, and has little melonworm to amplify band, can complete the detection to the medium and small melonworm of water body.
The collection water sample of described step 1 is the water sample equal-volume mixture of water sample or pool water port different time sections at the bottom of the required culturing pool pond detected, in pool wall different range.
Water sample in described step 2 is treated to and adopts 25 μm of filter membranes to carry out vacuum filtration to described water sample, after suction filtration, described filter membrane is put into the 50ml centrifuge tube that 35ml sterilized water is housed, concussion mixing on turbula shaker, the centrifugal 5min of 8000rpm, collecting precipitation is in-20 DEG C of preservations; Add new sterilized water to throw out, repeatedly after purge, be loaded in the centrifuge tube of 1.5ml, the centrifugal 5min of 10000rpm, abandons supernatant for subsequent use.
In described step 2, the extraction of STb gene adopts kit method, saves backup after detected through gel electrophoresis DNA extraction situation in-20 DEG C.
In described step 3, primer nucleotide sequences IC-ITS-F and IC-ITS-R is respectively IC-ITS-F5'-GTTCCCCTTGAACGAGGAATTC-3' and IC-ITS-R5'-TTAGTTTCTTTTCCTCCGC-3 '.Pcr amplification condition is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, 30 circulations, and 72 DEG C extend 10min;
Pcr amplification reaction system is in every 25 μ l systems: 10xbuffer2.5 μ l, MgCl
2(25mM) 3 μ l, dNTPs (2.5mM) 2 μ l, concentration is each 0.5 μ l of the positive anti-primer of 100pmol/ μ l, template DNA 1 μ l, Taq enzyme (5U/ μ l) 0.25 μ l, ddH
2o15.25 μ l;
The above-mentioned PCR primer of electrophoresis detection is in the sepharose of 1%, and voltage 120V electrophoresis 15min, finally takes pictures and can complete electrophoresis detection on gel imaging system.Chao Shi PCR primer for little melonworm is 2 right, is respectively n-IC1-F and n-IC1-R; N-IC2-F and n-IC1-R.Pair of primers (n-IC1-F and n-IC1-R) increases PCR primer 447bp after testing, and the second pair of primer (n-IC2-F and the n-IC1-R) PCR primer that increases is 225bp.
Individual 20-40 of the mono-clonal of picking in described step 4, finally order-checking obtains sequence is 15.
In described step 6, Chao Shi PCRPCR amplification condition is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, 30 circulations, and 72 DEG C extend 10min;
Pcr amplification reaction system is in every 25 μ l systems: 10xbuffer2.5 μ l, MgCl
2(25mM) 3 μ l, dNTPs (2.5mM) 2 μ l, concentration is each 0.5 μ l of the positive anti-primer of 100pmol/ μ l, template DNA 1 μ l, Taq enzyme (5U/ μ l) 0.25 μ l, ddH
2o15.25 μ l;
Environmental molecules biology techniques principle is applied to cultivation investigation by the present invention, when obtaining little melonworm DNA, only need in target water body certain limit, carry out water sampling in the time, fast, easy, understood the medium and small melonworm of water body timely there is situation, solve the problem of ichthyophthiriasis Feed Discovery difficulty, for laying the foundation eventually through water body little melonworm DNA concentration measuring and calculating ichthyophthiriasis outburst probability, to solve problem difficult due to the treatment that is in a bad way when ichthyophthiriasis finds.Relative to the prevention and controls of the little parasitosis of tradition, there is novelty, avoid the drug residue that drug treatment brings, the poor effect of physical therapy modalities, the problem that the Biotherapy method time is long, prevents trouble before it happens, greatly reduces the outburst probability of ichthyophthiriasis.
Accompanying drawing explanation
Fig. 1 is the method flow diagram of the embodiment of the present invention.
The DNA extraction result figure that Fig. 2 is the inventive method sensitivity technique, natural water breaks out little melonworm process, the little melonworm of different breeding water body exists situation.
Fig. 3 is that the embodiment of the present invention uses IC-ITS-F and IC-ITS-R to carry out the result figure of first time PCR to the little melonworm DNA in above-mentioned three kinds of situations.
Fig. 4 is that the embodiment of the present invention uses n-IC1-F and n-IC1-R to the result figure of the little melonworm first time PCR primer Chao Shi PCR in above-mentioned three kinds of situations.
Fig. 5 is that the embodiment of the present invention uses n-IC2-F and n-IC1-R to the result figure of the little melonworm first time PCR primer Chao Shi PCR in above-mentioned three kinds of situations.
Fig. 6 is the sequence of little melonworm and the sibling species obtained after universal primer ITS-F/ITS-R first time PCR, and in figure, box indicating is 2 couples of Auele Specific Primer (n-IC1-F/n-IC1-R for little melonworm specific site design; N-IC2-F/n-IC1-R); Wherein, Fig. 6-1n-IC1-F is upstream primer, 6-2n-IC1-R is downstream primer; Fig. 6-3n-IC2-F is upstream primer, 6-4n-IC1-R is downstream primer.
Embodiment
In order to more clearly understand technology contents of the present invention, below specific embodiment of the invention method is described further.
Accompanying drawing 2, 3, 4, 5 electrophorogram swimming lane point sample orders are followed successively by: the little melonworm of 5/L, the little melonworm of 10/L, the little melonworm of 15/L, the little melonworm of 25/L, the little melonworm of 40/L, make-up water source, copper fish, blank, DNAMarker, first stage water sample, subordinate phase water sample, phase III water sample, fourth stage water sample, five-stage water sample, blank, DNAMarker, adopt and feed No. 6, workshop pond water sample, No. 1, tail beach pool water sample, No. 3, tail beach pool water sample, board house glass cylinder culturing pool water sample, 4th No. 5, workshop pond water body, blank, DNAMarker.
DNAMrker stripe size is followed successively by from the bottom up: 100bp; 250bp; 500bp; 750bp; 1000bp; 2000bp; 3000bp; 5000bp.
Accompanying drawing 6 is for what obtain after universal primer ITS-F/ITS-R first time PCR, and the sequence of little melonworm and sibling species, totally 10, its sequence is shown in SEQUENCELISTING.
Embodiment 1
Method sensitivity technique
In order to determine that the present invention effectively can extract the little melonworm DNA in water body, get the fish body (there is a lot of small particles fish surface) of the little melonworm of severe infections, with distilled water clean fish body three times, with the white packing of slide glass scraping fish surface gently, packing is collected in sterile petri dish together with the ichthyophthirius multifiliis in it, leaves standstill, draw a large amount of mucus in another sterile petri dish and repeatedly purge, the little melonworm be wrapped in mucus is separated, removing mucus.So far there is the single polypide of little melonworm (if the more aforesaid operations that can again repeat of mucus is to obtain the independent polypide of more) can chosen from a large number in 2 culture dish.By 5, 10, 15, 25, 40 little melonworms are placed with 1L water respectively, test by experiment flow as shown in Figure 1, use filter sizes is that the vacuum air pump of 25 μm carries out suction filtration process to 5 water samples, filter membrane is put into the centrifuge tube of 50ml, concussion mixing on turbula shaker, the centrifugal 5min of 8000rpm, remove supernatant, add new sterilized water, repeatedly be sucked into after purge in the centrifuge tube of 1.5ml, the centrifugal 5min of 10000rpm, remove supernatant, the test kit using TIANGEN company to produce extracts little melonworm STb gene and is dissolved in TE damping fluid,-20 DEG C save backup.Fig. 2-A is DNA agarose gel electrophoresis detected result.
Carry out detected through gel electrophoresis to PCR primer after adopting IC-ITS-F and IC-ITS-R sequence to increase as versatility primer, result is as shown in Fig. 3-A.Above-mentioned PCR primer is cut glue to reclaim, proceed in bacillus coli DH 5 alpha after connection and clone, picking mono-clonal individuality checks order.DNAMAN6.0 software is utilized to compare, search with little melonworm difference site (search procedure in difference site as shown in Figure 6,10 sequences comprising little melonworm are obtained by universal primer IC-ITS-F and IC-ITS-R, through the comparison of DNAMAN6.0, voluntary election difference site is if the sequence in square frame is as Chao Shi PCR primer, wherein, Fig. 6-1,6-2 are respectively 5 ', 3 ' the section sequence of pair of primers n-IC1-F, n-IC1-R; Fig. 6-3,6-4 are respectively 5 ', 3 ' the section sequence of second couple of primer n-IC2-F, n-IC1-R.), design Auele Specific Primer n-IC1-F and n-IC1-R for little melonworm DNA; N-IC2-F and n-IC1-R.Utilize above-mentioned 2 pairs of Auele Specific Primers to carry out Chao Shi PCR, result is as shown in Fig. 4-A and Fig. 5-A.
Result shows, even under the polypide concentration of 5, the inventive method still well can detect the DNA fragmentation of little melonworm polypide.
In addition, this process of the test with the addition of institute's cultivation water source as a supplement the healthy copper fish of water sample (being namely respectively the 6th hole sample of Fig. 2-A, the 6th hole sample of 3-A, the 6th hole sample of 4-A, the 6th hole sample of 5-A) and a tail skin histology (being namely respectively the 7th hole sample of Fig. 2-A, the 7th hole sample of 3-A, the 7th hole sample of 4-A, the 7th hole sample of 5-A) in contrast, result shows these 2 samples all without little melonworm DNA, tentatively judges that institute cultivation water source and the healthy copper fish of this tail are without little melonworm.
Embodiment 2
Natural water breaks out little melonworm process
Aquarium sterilization as shown in Figure 1, is cleaned, is put into 2 tail copper fishes, be designated as the first stage by experiment flow; The Limnodrilus hoffmeisteri postscript that starts for second day to throw something and feed is subordinate phase; Slow in action at fish body, actively do not take food, fish body has fragmentary single white point to be designated as the phase III; Treat that fish surface exists more obvious white point and is designated as fourth stage; Treat fish body abdomeinal fin surface, gill portion white in flakes and fish body almost stationary state thing be designated as five-stage.Adopt siphonage at the bottom of the pond of culturing jar in these 5 stages respectively, water sample 1L is taked in the mixing of pool wall different positions, use filter sizes is that the vacuum air pump of 25 μm carries out suction filtration process to water sample, filter membrane is put into the centrifuge tube of 50ml, concussion mixing on turbula shaker, the centrifugal 5min of 8000rpm, remove supernatant, add new sterilized water, repeatedly be sucked into after purge in the centrifuge tube of 1.5ml, the centrifugal 5min of 10000rpm, remove supernatant, the test kit using TIANGEN company to produce extracts the total environment DNA in filter membrane and is dissolved in TE damping fluid,-20 DEG C save backup.Fig. 2-B is DNA agarose gel electrophoresis detected result.
Carry out detected through gel electrophoresis to PCR primer after adopting IC-ITS-F and IC-ITS-R sequence to increase as versatility primer, result is as shown in Fig. 3-B.Adopt Auele Specific Primer n-IC1-F and n-IC1-R; N-IC2-F and n-IC1-R carries out Chao Shi PCR respectively, and result is as shown in Fig. 4-B and Fig. 5-B.
Result shows, and infects 3 different time stages i.e. (three to the five-stage) of little melonworm initial stage to severe infections, all can amplify little melonworm DNA fragmentation from water body, and band brightness value is all different at fish body.
Embodiment 3
There is situation in the little melonworm of different breeding water body
Experiment flow as shown in Figure 1,5 different breeding places are chosen in mandarin sturgeon institute, carry out water body detection, result, as shown in Fig. 2-C, 3-C, 4-C, 5-C, represents respectively to adopt and feeds pond, No. 6, workshop, No. 1 pool, No. 3 pools, board house glass cylinder culturing pool, the water body situation in the 4th No. 5 ponds, workshop, result display only has board house glass cylinder storage pond, holding pond to there is a small amount of little melonworm DNA, and other water bodys are all without little melonworm.Investigation checking is except board house glass cylinder culturing pool is wild copper fish foster temporarily, other culturing pools cultivation object is sturgeon, sturgeon belongs to the species of the little melonworm of not easy infection because of own biological feature, so, supplementary water sample, the embodiment 2 of 1 can tentatively be learnt in conjunction with the embodiments, the cultivation water source of mandarin sturgeon institute there is not little melonworm, support the water body of copper fish temporarily and after feeding Limnodrilus hoffmeisteri, break out the fish body of little melonworm, cause of disease may be carried due to wild fish body itself or the Limnodrilus hoffmeisteri of throwing something and feeding, cause ichthyophthiriasis to break out when storage pond, holding pond environment is suitable for.
。
Claims (9)
1. a method for the medium and small melonworm of rapid detection water body, is characterized in that, comprise the following steps:
Step 1: according to Cultivated water size, gathers water sample;
Step 2: after being processed by water sample, extracts the STb gene in water body;
Step 3: utilize nucleotide sequence IC-ITS-F and IC-ITS-R to carry out pcr amplification as primer, obtain PCR primer 750bp, electrophoresis detection;
Step 4: above-mentioned PCR primer is cut glue and reclaim, proceed in bacillus coli DH 5 alpha after connection carrier and clone, and picking mono-clonal individuality as much as possible checks order;
Step 5: utilize DNAMAN6.0 software to compare to sequencing result, finds out the difference site between little melonworm and the sibling species recorded and designs at least 2 to the Chao Shi PCR primer for little melonworm;
Step 6: the PCR primer utilizing above-mentioned primer pair step 3 to obtain carries out Chao Shi PCR, knows that electrophoresis detection result shows, and has little melonworm to amplify band, can complete the detection to the medium and small melonworm of water body.
2. the method for the medium and small melonworm of rapid detection water body according to claim 1, it is characterized in that, the collection water sample of described step 1 is the water sample equal-volume mixture of water sample or pool water port different time sections at the bottom of the required culturing pool pond detected, in pool wall different range.
3. the method for the medium and small melonworm of rapid detection water body according to claim 1, it is characterized in that, water sample in described step 2 is treated to and adopts 25 μm of filter membranes to carry out vacuum filtration to described water sample, after suction filtration, described filter membrane is put into the 50ml centrifuge tube that 35ml sterilized water is housed, concussion mixing on turbula shaker, the centrifugal 5min of 8000rpm, collecting precipitation is in-20 DEG C of preservations; Add new sterilized water to throw out, repeatedly after purge, be loaded in the centrifuge tube of 1.5ml, the centrifugal 5min of 10000rpm, abandons supernatant for subsequent use.
4. the method for the medium and small melonworm of rapid detection water body according to claim 1, is characterized in that, in described step 2, the extraction of STb gene adopts kit method, saves backup after detected through gel electrophoresis DNA extraction situation in-20 DEG C.
5. the method for the medium and small melonworm of rapid detection water body according to claim 1, it is characterized in that, in described step 3, primer nucleotide sequences IC-ITS-F and IC-ITS-R is respectively IC-ITS-F5'-GTTCCCCTTGAACGAGGAATTC-3' and IC-ITS-R5'-TTAGTTTCTTTTCCTCCGC-3 '.
6. the method for the medium and small melonworm of rapid detection water body according to claim 1, it is characterized in that, in described step 3, pcr amplification condition is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations, 72 DEG C extend 10min;
Pcr amplification reaction system is in every 25 μ l systems: 10xbuffer2.5 μ l, MgCl
2(25mM) 3 μ l, dNTPs (2.5mM) 2 μ l, concentration is each 0.5 μ l of the positive anti-primer of 100pmol/ μ l, template DNA 1 μ l, Taq enzyme (5U/ μ l) 0.25 μ l, ddH
2o15.25 μ l;
The above-mentioned PCR primer of electrophoresis detection is in the sepharose of 1%, and voltage 120V electrophoresis 15min, finally takes pictures and can complete electrophoresis detection on gel imaging system.
7. the method for the medium and small melonworm of rapid detection water body according to claim 1, is characterized in that, the Chao Shi PCR primer for little melonworm is 2 right, is respectively n-IC1-F and n-IC1-R; N-IC2-F and n-IC1-R.
8. the method for the medium and small melonworm of rapid detection water body according to claim 1, is characterized in that, individual 20-40 of the mono-clonal of picking in described step 4.
9. the method for the medium and small melonworm of rapid detection water body according to claim 1, is characterized in that, in described step 6, Chao Shi PCRPCR amplification condition is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations, and 72 DEG C extend 10min;
Pcr amplification reaction system is in every 25 μ l systems: 10xbuffer2.5 μ l, MgCl
2(25mM) 3 μ l, dNTPs (2.5mM) 2 μ l, concentration is each 0.5 μ l of the positive anti-primer of 100pmol/ μ l, template DNA 1 μ l, Taq enzyme (5U/ μ l) 0.25 μ l, ddH
2o15.25 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610024984.6A CN105524996B (en) | 2016-01-15 | 2016-01-15 | A kind of method of small melonworm in quick detection water body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610024984.6A CN105524996B (en) | 2016-01-15 | 2016-01-15 | A kind of method of small melonworm in quick detection water body |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105524996A true CN105524996A (en) | 2016-04-27 |
| CN105524996B CN105524996B (en) | 2019-05-10 |
Family
ID=55767519
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610024984.6A Active CN105524996B (en) | 2016-01-15 | 2016-01-15 | A kind of method of small melonworm in quick detection water body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105524996B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868120A (en) * | 2017-02-14 | 2017-06-20 | 中国水产科学研究院黄海水产研究所 | Virulence gene is the rapid screening method of the Escherichia coli of Stx1 in fresh water |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103866020A (en) * | 2014-03-13 | 2014-06-18 | 厦门出入境检验检疫局检验检疫技术中心 | Primer, kit and method for detecting Ichthyophthirius multifiiis |
-
2016
- 2016-01-15 CN CN201610024984.6A patent/CN105524996B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103866020A (en) * | 2014-03-13 | 2014-06-18 | 厦门出入境检验检疫局检验检疫技术中心 | Primer, kit and method for detecting Ichthyophthirius multifiiis |
Non-Patent Citations (2)
| Title |
|---|
| OLIVIER JOUSSON等: "Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR", 《DISEASES OF AQUATIC ORGANISMS》 * |
| 赵增连等: "《观赏鱼鉴赏与检疫指南》", 31 August 2014 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106868120A (en) * | 2017-02-14 | 2017-06-20 | 中国水产科学研究院黄海水产研究所 | Virulence gene is the rapid screening method of the Escherichia coli of Stx1 in fresh water |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105524996B (en) | 2019-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pérez‐Ortega et al. | Symbiotic lifestyle and phylogenetic relationships of the bionts of Mastodia tessellata (Ascomycota, incertae sedis) | |
| Lollis et al. | Molecular characterization of Histomonas meleagridis and other parabasalids in the United States using the 5.8 S, ITS-1, and ITS-2 rRNA regions | |
| CN104178490B (en) | For the cereal cyst nematode RNAi site sequences and its carrier of biological prevention and control and application | |
| CN102134593B (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| Meng et al. | The characterization, expression and activity analysis of superoxide dismutases (SODs) from Procambarus clarkii | |
| Karpov et al. | Gromochytrium mamkaevae gen. & sp. nov. and two new orders: Gromochytriales and Mesochytriales (Chytridiomycetes) | |
| CN104498599B (en) | One group of microsporidian molecule universal detector primer and kit thereof | |
| CN104630369B (en) | The PCR detection method of fusarium moniliforme | |
| Kim et al. | The marine dinoflagellate genus Dinophysis can retain plastids of multiple algal origins at the same time | |
| CN102220247B (en) | Glycyrrhiza endophytic fungi for producing glycyrrhetic acid | |
| CN105296479B (en) | A kind of specific primer of mulberry tree bacterial wilt identification and its application | |
| CN102643756B (en) | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice | |
| KR101615433B1 (en) | SNP marker for identifying the antlered form Ganoderma lucidum, and identifying method using the same | |
| CN103789441B (en) | SSR (Simple Sequence Repeat) molecular marker method for identifying two hippocampus populations | |
| CN105524996A (en) | Method for rapid detection of ichthyophthirius multifiliis in water body | |
| CN104789691A (en) | Litopenaeus vannamei genetic sex identification method based on one-step PCR (polymerase chain reaction) technique | |
| CN103361340B (en) | Bay scallop thermostable related heat shock protein 70 gene marker and assistant breeding method thereof | |
| CN102115740B (en) | Method for extracting genomic DNA (Deoxyribose Nucleic Acid) from fish myxosporean | |
| CN104498593B (en) | Identify or auxiliary identify storage bean weevil primer to and test kit | |
| Sørensen et al. | Ichthyodinium identified in the eggs of European eel (Anguilla anguilla) spawned in captivity | |
| CN104962660A (en) | Ruditapes philippinarum species real-time fluorescent PCR (polymerase chain reaction) specific detection system and application thereof | |
| CN103789305A (en) | Molecular identification method for gene type of gold-silver feather locus of chicken | |
| CN105567827B (en) | PCR method that is a kind of while detecting a variety of Myxosporidia From Freshwaters of hybridized prussian carp | |
| CN106868134B (en) | Real-time quantitative PCR detection method for spores/gametes of ulva aquatica, ulva scleoides and hemerocallis fulva in sea cucumber culture water | |
| Lizarraga et al. | Incidents of snake fungal disease caused by the fungal pathogen Ophidiomyces ophidiicola in Texas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |